PROJECT SUMMARY The cardiomyocyte contractile apparatus is central to heart function. Mutations in the genes encoding sarcomere proteins like myosin heavy chain and myosin binding protein-C cause inherited forms of cardiomyopathy and an increased risk for cardiac arrhythmias. However, the mechanisms by which sarcomere protein mutations lead to arrhythmias remains largely unknown. We identified a novel component of the myofilament, myosin binding protein H-like (MyBP-HL). Myosin binding protein H was originally discovered along with myosin binding protein C. We found that MyBP-H is encoded by two distinct genes, MYBPH which specifies H-protein in skeletal muscle, and MYBPHL, which generates the H-protein of the heart. Moreover, we discovered that MYBPHL is highly enriched in the atria and expressed throughout the ventricle in a pattern consistent with ventricular conduction system cells. We identified a premature stop variant in MYBPHL (R255X) in a family with dilated cardiomyopathy and atrial and ventricular arrhythmias. Deletion of Mybphl in mice recapitulates this human phenotype, including atrial and ventricular arrhythmias and dilated cardiomyopathy. Despite low-level expression of MYBPHL in the left ventricle, heterozygous mutations of MYBPHL in mice and humans leads to left ventricular dysfunction. These observations suggest that MyBP-HL regulates myofilament content in the cardiac conduction system, an understudied area with regard to myofilament content and regulation. We hypothesize that MyBP-HL regulates sarcomere size and contractility and contributes to the function and morphology of ventricular conduction cells, and that loss of MyBP-HL leads to structural changes of these cells which, in turn, promotes arrhythmias and left ventricular dysfunction. We propose to study Mybphl in ventricular conduction cells by crossing the Mybphl null mouse with a conduction system reporter mouse and by creating a conditional Mybphl null mouse line for deletion of Mybphl in the adult heart and in the ventricular conduction system specifically. Dr. David Barefield, the PI of this project, has a strong background studying myofilament proteins and mouse models of cardiomyopathy. The training proposed in this study will allow Dr. Barefield to study mouse models of arrhythmia in order to establish the role of MYBPHL in regulating the ventricular conduction system. This will be done by collaborating with experts in techniques for studying whole-animal, whole-heart, and cellular electrophysiology. The candidate is taking the steps to become an independent academic investigator with his own laboratory at a university or medical research center in the United States. The exceptional environment and commitment to this research at Northwestern University, in addition to the expert team of co-mentors provides an outstanding environment to achieve the goals set out in this proposal.